A Genome-Wide Identification and Expression Pattern of LMCO Gene Family from Turnip (Brassica rapa L.) under Various Abiotic Stresses

Laccase-like multi-copper oxidases (LMCOs) are a group of enzymes involved in the oxidation of numerous substrates. Recently, these enzymes have become extremely popular due to their practical applications in various fields of biology. LMCOs generally oxidize various substrates by linking four-electron reduction of the final acceptor, molecular oxygen (O2), to water. Multi-copper oxidases related to laccase are extensively distributed as multi-gene families in the genome sequences of higher plants. The current study thoroughly investigated the LMCO gene family (Br-Lac) and its expression pattern under various abiotic stresses in B. rapa L. A total of 18 Br-Lac gene family members located on five different chromosomes were identified. Phylogenetic analysis classified the documented Br-Lac genes into seven groups: Group-I (four genes), Group-II (nine genes), Group-III (eight genes), Group-IV (four genes), Group-V (six genes), and Group-VI and Group-VII (one gene each). The key features of gene structure and responsive motifs shared the utmost resemblance within the same groups. Additionally, a divergence study also assessed the evolutionary features of Br-Lac genes. The anticipated period of divergence ranged from 12.365 to 39.250 MYA (million years ago). We also identified the pivotal role of the 18 documented members of the LMCO (Br-lac) gene family using quantitative real-time qRT-PCR. Br-Lac-6, Br-Lac-7, Br-Lac-8, Br-Lac-16, Br-Lac-17, and Br-Lac-22 responded positively to abiotic stresses (i.e., drought, heat, and salinity). These findings set the stage for the functional diversity of the LMCO genes in B. rapa.


Introduction
Multi-copper oxidases (MCOs) are a group of enzymes from the kingdom Fungi that contain laccases and peroxidases [1,2], metal oxidases from bacteria [3], and ascorbate oxidases from the kingdom Plantae [4]. LMCOs, commonly known as "laccases", are mainly involved in the oxidation of industrial substrates as well as in developmental processes in plants. Some of the prominent developmental processes include polymerization of lignin and flavonoids and anthocyanin degradation. These are very helpful in the browning of the pericarp during postharvest in litchi fruit [5]. Lignin digestion in fungi [6], metabolic activities (e.g., pigment production), and several other activities, such as pathogenic virulence in bacteria and plant species, are other key features of these enzymes [7,8]. Metal transportation and homeostasis in bacterial cells [9,10], healing of wounds, and lignin production in higher plants are also associated with laccases [11,12].

Classification of A. thaliana and B. rapa LMCO Genes
A maximum likelihood (ML) phylogenetic tree was constructed for the 35 reported LMCO genes (i.e., 17 from A. thaliana (At-Lac) and 18 from B. rapa (Br-Lac)). The ML phylogenetic tree divided all the documented 35 LMCO genes into seven groups (Table S1).
The protein lengths of the Br-Lac genes from B. rapa ranged from 558 to 582 amino acids (aa). The molecular weights of the resultant proteins were between 61.22 and 65.75 kDa, while pIs varied from 6.07 to 9.73. These proteins possess a negative grand average of hydropathicity (GRAVY), indicating hydrophilic behavior. Understanding plant functions requires knowledge of subcellular localization, and our results showed that most of the proteins were found in the chloroplast and vacuoles (Table 1).

Classification of A. thaliana and B. rapa LMCO Genes
A maximum likelihood (ML) phylogenetic tree was constructed for the 35 reported LMCO genes (i.e., 17 from A. thaliana (At-Lac) and 18 from B. rapa (Br-Lac)). The ML phylogenetic tree divided all the documented 35 LMCO genes into seven groups (Table  S1). Additionally, the exon-intron structures of the 35 LMCO genes were positioned according to their phylogenetic relationships. LMCO genes of the same group showed more similarities with the LMCO protein sequences. The exon and intron numbers differed between the LMCO genes of A. thaliana and those of B. rapa. The number of exons in the At-Lac and Br-Lac genes was five to seven in A. thaliana and B. rapa (Figure 3).

Conserved Motif Distribution
Fifteen conserved motifs were identified in A. thaliana and B. rapa LMCO proteins using the MEME tool and MEME suite web server to acquire the logos (

Conserved Motif Distribution
Fifteen conserved motifs were identified in A. thaliana and B. rapa LMCO protein using the MEME tool and MEME suite web server to acquire the logos (

Calculating the Non-Synonymous (Ka) and Synonymous (Ks) Substitution Rates
As part of the divergence analysis, the non-synonymous substitution p non-synonymous site (Ka) and synonymous substitution per synonymous site (Ks) f each pair of paralogous Br-Lac genes were calculated according to the phylogenetic tr server ( Figure S2). This was carried out to understand the degree of evolutionary discr tion among Br-Lac genes. A Ka/Ks value lower than 1 demonstrated the presence of p rifying selection pressure during evolution. Each pair of Br-Lac genes went through period of divergence approximately 12.365 to 39.250 million years ago ( Table 2).

Calculating the Non-Synonymous (Ka) and Synonymous (Ks) Substitution Rates
As part of the divergence analysis, the non-synonymous substitution per non-synonymous site (Ka) and synonymous substitution per synonymous site (Ks) for each pair of paralogous Br-Lac genes were calculated according to the phylogenetic tree server ( Figure S2). This was carried out to understand the degree of evolutionary discretion among Br-Lac genes. A Ka/Ks value lower than 1 demonstrated the presence of purifying selection pressure during evolution. Each pair of Br-Lac genes went through a period of divergence approximately 12.365 to 39.250 million years ago (Table 2).

Protein Structure Analysis of Br-Lac Genes
To obtain a more in-depth comprehension of the framework of the Br-Lac genes, the secondary and tertiary structures of the Br-Lac proteins were investigated (Table 3, Figure 5). Both folding and coiling helped in understanding the secondary structure of the Br-Lac proteins. The secondary structure of the Br-Lac proteins was made up of four basic components: the helix (H%), the turn (T%), the extended chain (E%), and the random coil (RC%). Following the helix (H%), which varied from 12.2% (Br-Lac-9) to 21.11% (Br-Lac-9), the random coil (RC%) in the secondary structure of Br-Lac proteins had the greatest value, ranging from 36.67% (Br-Lac-10) to 45.7% (Br-Lac9). The extended chain (E%) ranged from 28.77% (Br-Lac-3) to 32.99% (Br-Lac-1). The secondary structure of Br-Lac proteins was also supported by the tertiary structures of the proteins, as shown using ExPASy (https://swissmodel.expasy.org/) (accessed on 7 January 2023).

Expression Patterns of LMCO Genes Under Abiotic Stress
We exposed B. rapa seedlings to various abiotic stresses (drought, salinity, and heat) to expose the expression patterns of its 18 documented LMCO genes. QRT-PCR was performed on the 18 Br-Lac genes at different time intervals after various abiotic treatments, and the expression levels were calculated ( Figure 5). The expression patterns of the 18 Br-Lac genes showed transcriptional changes under abiotic stress. It was concluded that members of the LMCO gene family show a response to multiple stresses.

Expression Patterns of LMCO Genes under Abiotic Stress
We exposed B. rapa seedlings to various abiotic stresses (drought, salinity, and heat) to expose the expression patterns of its 18 documented LMCO genes. QRT-PCR was performed on the 18 Br-Lac genes at different time intervals after various abiotic treatments, and the expression levels were calculated ( Figure 5). The expression patterns of the 18 Br-Lac genes showed transcriptional changes under abiotic stress. It was concluded that members of the LMCO gene family show a response to multiple stresses. Five genes (Br-Lac-1, Br-Lac-2, Br-Lac-6, Br-Lac-7, and Br-Lac-8) of the 18 Br-Lac genes were noted to have low expression levels in response to drought stress. At different time intervals, the expression levels of these five genes were down-regulated, while four genes (Br-Lac-4, Br-Lac-5, Br-Lac-16, and Br-Lac-17) were up-regulated. The expression level of Br-Lac-5 was increased at 12 h and 24 h but suddenly reversed at 48 h and 72 h. After drought treatment, the up-regulated genes (Br-Lac-4, Br-Lac-5, Br-Lac-16, and Br-Lac-17) may be related to drought tolerance ( Figure 6). stress treatment. At the same time, only three genes were down-regulated under h stress conditions (Br-Lac-2, Br-Lac-6, and Br-Lac-7). Noteworthy, our findings sugg that the expression levels of Br-Lac-4, Br-Lac-9, and Br-Lac-16 were induced by droug heat, and salinity stresses, suggesting that these genes might be important for resista to abiotic stresses. A list of the primers is given in Table S2.  . Noteworthy, our findings suggest that the expression levels of Br-Lac-4, Br-Lac-9, and Br-Lac-16 were induced by drought, heat, and salinity stresses, suggesting that these genes might be important for resistance to abiotic stresses. A list of the primers is given in Table S2.

Discussion
Determining the physiological functions of LMCOs is comparatively difficult due to their wide distribution and complex nature across various plant species. These factors, in combination with several others, make it very challenging to identify LMCO genes and their functions. Differentiating the xylem of Liriodendron tulipifera, Pinus taeda, Zinnia elegans, and Acer pseudoplatanus was used to isolate these enzymes [19][20][21][22]. Lignin monomers were oxidized in vitro by these enzymes, obtained from Pseudoplatanus cells, when maintained in suspension culture [23]. Prototype LMCOs (laccase) from Rhus vernicifera were found to be good in healing wounds caused either by herbivores or in response to pathogens [20,24]. The trunks of Japanese lacquer and Rhus vernicifera were reported to exhibit an oxidative polymerization reaction between laccase and the alkyl catechols in latex sap, causing a strong protective seal to form over the injury. This happens because laccase is a component of latex sap [20,25]. Furthermore, biochemical evidence also supports the idea that plant LMCOs play a role in iron metabolism [21].
A genome-wide gene family study is the initial step in understanding gene structure, function, and evolution [26]. Additionally, sequence-based searching and phylogenetic characterization are the most efficient techniques for identifying laccase genomes [27][28][29][30][31]. We carried out a thorough search for LMCO genes across the B. rapa genome and found a total of 18 genes. Based on domain organization and evolutionary analyses, these genes were further classified into seven subgroups (Figure 1). According to a synteny study, B. rapa and A. thaliana LMCO genes have significant similarities (Figures 3 and 4). These results are remarkably consistent with those of earlier studies [20,32].
Isoelectric focusing points (pIs) have been used as the primary classification method for plant LMCOs, with the implicit belief that pI may be related to substrate kinetics and enzyme activity [6,20]. A search for the conserved domain, motif, and structure was carried out to better understand the shared characteristics and biological roles of LMCO genes (Figures 3 and 4). The persistence of ancient and more modern gene duplication events showed that both purifying and diversifying selection lead to the emergence of new gene functions over time [32][33][34][35]. The current work identified and analyzed 18 LMCO genes in B. rapa and 17 LMCO genes in A. thaliana.
Biotic and abiotic stresses define the patterns of both gene expression and function [36]. QRT-PCR has been used in several research studies to investigate transcript levels, such as the expression profile of At-Lac-12 and At-Lac-14 in response to abiotic stresses. A. thaliana LMCO proteins were successfully involved in the process of drought tolerance [37]. To obtain further insights into the regulation of Br-Lac in response to abiotic stress, gene expressions in B. rapa were analyzed using real-time RT-PCR, as shown in  [38] reported that expressions of the At-Lac-12 and At-Lac-14 genes were very limited in heat and drought stress conditions. In another study, At-Lac-17 and At-Lac-22 overexpression promoted drought resistance in A. thaliana, whereas the expression levels of At-Lac-9, At-Lac-11, and At-Lac-15 were inhibited by drought and salinity stress.
There has only been relatively little research on the LMCO gene family in plants. The LMCO gene family's intron-exon configurations may cause functional variability. Such findings could be attributable to a homology structure separate from the domain sequences [21,33]. This study established a baseline for understanding the molecular function and response to abiotic stresses of Br-Lac genes and recommends future research for examining these genes concerning various biological functions.

Phylogenetic Analysis
A phylogenetic tree for the full-length LMCO protein sequences of A. thaliana and B. rapa was constructed using MEGA software version 7.0. [42]. The ClustalW program was used to align the full-length LMCO protein sequences. The pairwise deletion option was selected, and the Poisson model with a 1000 bootstrap sample was used by applying the maximum likelihood method to make an LMCO tree.

Localization of the Chromosomes and Synteny Analysis
We examined genome data for the location of LMCO genes on the B. rapa chromosome. Syntenic relationships in the Brassica genomes were constructed using a method similar to the Plant Genome Duplication Database (PGDD; http://chibba.agtec.uga.edu/ duplication/) (accessed on 28 December 2022) [43]. The syntenic link between the genes of the LMCO family was examined using TBtools for the investigation of evolutionary history [44].

Protein Physicochemical Properties of LMCO
The NCBI database (https://www.ncbi.nlm.nih.gov/) (accessed on 7 January 2022) was used to determine amino acid (bp), CDS (bp), and location on the chromosome of the LMCO gene to clarify its physicochemical characteristics. Isoelectric points (pI), molecular weight (MW), grand average of hydropathicity (GRAVY), and the formula of the individual LMCO gene in B. rapa were assessed through the ProtParam tool on the ExPASy server (http: //web.expasy.org/protparam) (accessed on 29 April 2023). We also used the WoLF PSORT service for plant protein location (https://wolfpsort.hgc.jp/) (accessed on 29 April 2023) to assess LMCO protein in Brassica [49].

Calculating Ka and Ks
The substitution rates of Ka and Ks of the syntenic gene pairs were measured with Nei-Gojobori using TBtools software. We used the KaKs calculator 2.0 with the Nei-Gojobori method, as practiced by several workers [48][49][50].

Plant Materials and Stress Treatments
B. rapa seeds were soaked in water (2 days) for the process of germination. The soaked seeds were then cultured in 10 cm plastic pots. The germinated seedlings were then allowed to grow in a chamber room under light and dark (16/8) at 25 • C until the four-leaf stage. Different abiotic stresses (i.e., drought, heat, and salinity) were applied to the growing seedlings [51]. For drought conditions, the germinated seedlings were treated with 20% polyethylene glycol-6000 (PEG-6000), while 200 mM NaCl was used to initiate salinity stress. For heat stress, the germinated seedlings were exposed to a high temperature of 40 • C. To calculate the seedlings' response to drought stress, we collected leaf samples after 3, 6, 12, 24, 48, and 72 h [52]. In a similar pattern, young leaves were collected after 3, 6, 12, 24, and 48 h to document resistance to salinity stress. After collection, the leaf samples were kept in liquid nitrogen and stored at −80 • C for RNA isolation.

RNA Extraction and Real-Time qRT-PCR
A Plant Total RNA Isolation Kit (FOREGENE, China) was used for total RNA extraction from differently treated samples (i.e., drought, salinity, or heat). A RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, (Waltham, Massachusetts, USA) was used to synthesize purified cDNA with 1 µg of total RNA and oligo primers [53]. Light Cycler ® 480 II (Mannheim, Roche, Germany) and SYBR Green I Master Mix (Roche, Germany) were used to perform qPCR. The gene-specific primers were designed using the NCBI online software primer designing tool (https://www.ncbi.nlm.nih.gov/tools/ primer-blast/) (accessed on 15 January 2023) (Table S2). Each qPCR reaction was conducted as follows: 0.2 µL of cDNA, 5 µL of 0.5 µM gene-specific primer pre-mixture, 10 µL of 2 × SYBR Green Master Mix, and 4.8 µL of water. Actin7 was used as the internal standard to normalize the expression levels for target genes. A melting curve was used to evaluate the specificity of amplification. All experiments had three biological replicates and technical replicates. The 2−∆CT method was used for data calculation. Graph Pad Prism 9 was used to analyze and graph the expression data.

Conclusions
In conclusion, 18 LMCO genes were found in B. rapa during this investigation. They were categorized into seven groups based on phylogenetic analysis and similarities in amino acid sequences. The basic gene parameters, such as amino acid and CDS length, molecular weight (MW/kDa), isoelectric point (PI), and GRAVY, as well as the fact that the majority of Br-Lac genes were located in chloroplasts, were unearthed by the physicochemical properties of Br-Lac genes. The divergence study further illuminated the Br-Lac genes' evolutionary history, which revealed that the time of divergence ranged from 12.365 to 39.250 MYA. These findings point to a shared biological function of Br-Lac genes in response to enzymatic activity and offer helpful hints for further research into the diversity and function of Br-Lac genes. Expression analyses of the Br-Lac gene family were performed in this study for abiotic stress. The findings suggest that Br-lac genes resist heat, drought, and salinity stress.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/plants12091904/s1, Figure S1. Fifteen conserved motifs were identified in Arabidopsis and Brassica LMCO proteins using the MEME tool and MEME suite web server to acquire the logos; Figure S2. Pair of paralogous Br-Lac genes according to the phylogenetic tree server; Table S1, Groups base on the phylogenetic tree and motifs analysis of Arabidopsis and Brassica LMCO genes; Table S2, List of LMCO genes primers for qRT-PCR. Data Availability Statement: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.